Proteolysis and bioconversion of cereal proteins to glutamate and γ-Aminobutyrate (GABA) in Rye malt sourdoughs

This study aimed to achieve the conversion of cereal proteins to the alternative end products glutamate or γ-aminobutyrate (GABA). Rye malt, fungal proteases, and lactobacilli were employed to convert wheat gluten or barley proteins. Glutamate and GABA formations were strain-dependent. Lactobacillus reuteri TMW1.106 and Lactobacillus rossiae 34J accumulated glutamate; L. reuteri LTH5448 and LTH5795 accumulated GABA. Glutamate and GABA accumulation by L. reuteri TMW1.106 and LTH5448 increased throughout fermentation time over 96 h, respectively. Peptides rather than amino acids were the main products of proteolysis in all doughs, and barley proteins were more resistant to degradation by rye malt proteases than wheat gluten. However, addition of fungal protease resulted in comparable degradation of both substrates. Glutamate and GABA accumulated to concentrations up to 63 and 90 mmol kg(-1) DM, respectively. Glutamate levels obtained through bioconversion of cereal proteins enable the use of hydrolyzed cereal protein as condiment.     

Isolation and identification of sea buckthorn (Hippophae rhamnoides) phenolics with antioxidant activity and α-glucosidase inhibitory effect

This study was performed to evaluate the antioxidant and α-glucosidase inhibitory effects from the extract, fractions, and isolated compounds of sea buckthorn leaves. Six compounds, kaempferol-3-O-β-D-(6'-O-coumaryl) glycoside, 1-feruloyl-β-D-glucopyranoside, isorhamnetin-3-O-glucoside, quercetin 3-O-β-D-glucopyranoside, quercetin 3-O-β-D-glucopyranosyl-7-O-α-L-rhamnopyranoside, and isorhamnetin-3-O-rutinoside, were isolated from sea buckthorn leaf extracts. The butanol fraction (EC(50) = 1.81 μg/mL) along with quercetin 3-O-β-D-glucopyranoside (EC(50) = 1.86 μg/mL) had a higher DPPH radical-scavenging activity and showed stronger reducing power (OD(700) = 1.83 and 1.78, respectively). The butanol fraction (477 mg GAE/g) contained the highest amount of phenolic compounds and also the most powerful α-glucosidase inhibitory effect (86%) at 5 μg/mL. The results indicate that sea buckthorn leaf extracts could potentially be used for food additives and the development of useful natural compounds.     

Studies of O,O-dimethyl α-(2,4-dichlorophenoxyacetoxy)ethylphosphonate (HW02) as a new herbicide. 1. Synthesis and herbicidal activity of HW02 and analogues as novel inhibitors of pyruvate dehydrogenase complex

On the basis of the previous work for optimization of O,O-diethyl α-(substituted phenoxyacetoxy)alkylphosphonates, further extensive synthetic modifications were made to the substituents in alkylphosphonate and phenoxy moieties of the title compounds. New O,O-dimethyl α-(substituted phenoxyacetoxy)alkylphosphonates were synthesized as potential inhibitors of pyruvate dehydorogenase complex (PDHc). Their herbicidal activity and efficacy in vitro against PDHc were examined. Some of these compounds exhibited significant herbicidal activity and were demonstrated to be effective inhibitors of PDHc from three different plants. The structure-activity relationships of these compounds including previously reported analogous compounds were studied by examining their herbicidal activities. Both inhibitory potency against PDHc and herbicidal activity of title compounds could be increased greatly by optimizing substituent groups of the title compounds. O,O-Dimethyl α-(2,4-dichlorophenoxyacetoxy)ethylphosphonate (I-5), which acted as a competitive inhibitor of PDHc with much higher inhibitory potency against PDHc from Pisum sativum and Phaseolus radiatus than from Oryza sativa , was found to be the most effective compound against broadleaf weeds and showed potential utility as herbicide.     

Purification and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei with high specific activity and its gene sequence

Production, purification, and characterization of a novel β-1,3-1,4-glucanase (lichenase) from thermophilic Rhizomucor miehei CAU432 were investigated. High-level extracellular β-1,3-1,4-glucanase production of 6230 U/mL was obtained when oat flour (3%, w/v) was used as a carbon source at 50 °C. The crude enzyme was purified to homogeneity with a specific activity of 28818 U/mg. The molecular weight of purified enzyme was estimated to be 35.4 kDa and 33.7 kDa by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature of the enzyme were pH 5.5 and 60 °C, respectively. The K(m) values of purified β-1,3-1,4-glucanase for barley β-glucan and lichenan were 2.0 mM and 1.4 mM, respectively. Furthermore, the gene (RmLic16A) encoding the β-1,3-1,4-glucanase was cloned and its deduced amino acid sequence showed the highest identity (50%) to characterized β-1,3-1,4-glucanase from Paecilomyces thermophila. The high-level production and biochemical properties of the enzyme enable its potential industrial applications.     

Effect of herbicide used with years (8 + 1) on soil enzymic activity and microbial population diversity

Purpose

Soil microbes play important roles in plant nutrition and soil conservation, and the diversity and population of soil microbe are influenced by abiotic and biotic factors associated with different soil managements. However, the information concerning soil microbe diversity and population structure and its relation with soil fertility and enzyme activities are scarce in crop rotation under different soil management system.

Materials and methods

This paper reports the effects of three weeding managements (herbicide (2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methylphenyl) acetomide, C1₄H₂₀ClNO₂), manual weeding, and no weeding (CK)), on soil microbial diversity, population structure, and its relationship with soil active organic matter (AOM) and pH, and the activity of soil enzymes like sucrase, catalase, and urease activities from long-term test area in red soil upland field in southeast China, which was set up since 2006. Soil samples at 0–20-cm depths were collected before (8 years) and after (8 + 1 years) weeding management in April 2014.

Results and discussion

Soil enzymes (sucrase, catalase, and urease activity) and soil microbial populations had no significant difference (P > 0.05) under the three weeding treatments. Based on richness of microbial population up to 0.10%, the phyla Proteobacteria and Actinobacteria highly dominated the three soil treatments, averagely accounting for 21.76 and 21.44%. Chloroflexi was the next phylum, about accounting for 6.84%. Firmicutes, Verrucomicrobia, and Planctomycetes phylum accounted for 4.98, 4.78, and 4.23%, respectively. The percentage of Gemmatimonadetes was 2.76%, and that of Bacteroidetes was about 1.45%. Armatimonade and Nitrospira were the lowest, with 0.69 and 0.26%, respectively. Among the 20 phyla, only 5 had significant correlation with some of the soil properties. Twenty-one in 46 classes had significant correlation with some of the soil properties. Armatimonadetes and Fusobacteria had positive correlation with moisture. Acidobacteria_Gp3, Deltaproteobacteria, Chthonomonadetes, Armatimonadetes_gp4, and Euryarchaeota also were positively correlated with moisture. Negative correlation between Armatimonadetes, Chloroflexi, Chthonomonadetes, and Armatimonadetes_gp5 and AOM exists, and Armatimonadetes, Chthonomonadetes, Clostridia, Armatimonadetes, and pH were negatively correlated. Fusobacteria was positively correlated with catalase. Acidobacteria_Gp10 and Armatimonadia were positively correlated with catalase. Chthonomonadetes, Clostridia, and Armatimonadetes_gp5 were correlated with urease. Gammaproteobacteria and Flavobacteria were correlated with sucrase.

Conclusions

For long-term herbicide experiment conducted on the Dongxiang upland site, no significant effect of herbicide on soil microbial community composition and enzyme activities was found. Further work is needed to relate microbial community structure and function in different herbicide systems or season sampling, even to detect herbicide effect on community structure during the growing season.

     

Identification of cyanidin 3-O-β-(6″-(3-hydroxy-3-methylglutaroyl)glucoside) and other anthocyanins from wild and cultivated blackberries

Anthocyanins from blackberries are natural dietary pigments. The aim of this study was to investigate the occurrence of anthocyanins in fruits of wild Norwegian blackberries and three blackberry ( Rubus fruticosus L.) cultivars and to report the complete identification of cyanidin 3-O-β-(6″-(3-hydroxy-3-methylglutaroyl)glucopyranoside), 5. This new pigment is most probably the same pigment that has previously been reported to occur in various blackberry samples as cyanidin 3-dioxalylglucoside. All of the examined blackberry samples contained in similar relative proportions the 3-glucoside (1), 3-rutinoside (2), 3-xyloside (3), and 3-O-β-(6″-malonylglucoside) (4) of cyanidin and 5. The absolute amounts of 1-5 in the wild Norwegian blackberries were 249, 18, 10, 24, and 22 mg of cyanidin 3-glucoside equivalents/100 g of fresh weight, respectively.     

Profiles and α-amylase inhibition activity of proanthocyanidins in unripe Manilkara zapota (chiku)

Proanthocyanidins in unripe Manilkara zapota (chiku) were isolated using solvent extraction followed by Sephadex LH-20 fractionation with a yield of 0.9%. HPLC analysis using a diol column revealed well-resolved oligomers ranging from dimer to hexamer. The majority of the proanthocyanidins are composed of higher-degree oligomers appearing as one large peak in the chromatogram. Analysis of the proanthocyanidins using LC/MS showed that (epi)gallocatechins were the dominant extension unit in the proanthocyanidins. In agreement with this result, thiolysis treatment of the proanthocyanidins using mercaptoacetic acid produced thioether derivatives of (epi)gallocatechins as the major product and (epi)gallocatechin gallate derivatives as the minor product. The mean of the degree of polymerization was estimated to be 9.0. From MALDI-TOF MS, B-type gallocatechin oligomers up to decamer could be detected. The unripe chiku proanthocyanidins are thus good starting material for preparation of (epi)gallocatechin derivatives. The proanthocyanidins was shown to inhibit α-amylase with an IC(50) value of 4.2 ± 0.2 μg/mL and inhibit α-glucosidase with an IC(50) of 16.6 ± 0.3 μg/mL.     

High-performance liquid chromatography with photodiode array detection (HPLC-DAD)/HPLC-mass spectrometry (MS) profiling of anthocyanins from Andean Mashua Tubers (Tropaeolum tuberosum Ruíz and Pavón) and their contribution to the overall antioxidant activity

Mashua (Tropaeolum tuberosum Ruíz and Pavón), an Andean tuber with high antioxidant activity, has sparked interest because of its traditional medicinal use. In this study, we evaluated the anthocyanin composition for three purple mashua genotypes and their contribution to the overall antioxidant activity of the tuber. Mashua anthocyanins, total phenolics, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) antioxidant activity ranged from 45.5 to 131.9 mg of cyanidin 3-glucoside equivalents/100 g fresh weight (FW), 174.9 to 275.5 mg of gallic acid equivalents/100 g of FW, and 16.2 to 45.7 micromol of Trolox equivalents/g of FW, respectively. The high-performance liquid chromatography with photodiode array detection (HPLC-DAD) and HPLC-electrospray ionization tandem mass spectrometry (ESI/MS-MS) profiles revealed the presence of 11 different anthocyanins. The two major pigments (56.4-73.0% total area range at 520 nm) were identified as delphinidin 3-glucoside-5-acetylrhamnoside and delphinidin 3-sophoroside-5-acetylrhamnoside. Other pigments were delphinidin 3-glucoside-5-rhamnoside, delphinidin 3-sophoroside-5-rhamnoside, delphinidin 3-glucoside, cyanidin 3-sophoroside, and cyanidin 3-sophoroside-5-rhamnoside. Cyanidin 3-glucoside and cyanidin 3-rutinoside were only found in two genotypes, while pelargonidin 3-sophoroside and pelargonidin 3-sophoroside-5-rhamnoside were only found in the third one. Anthocyanins from mashua were the major contributors to the total ABTS values for only one of the three genotypes, suggesting that other phenolics present are playing a major role in the antioxidant power of mashua tubers. Results from this study provide important information for the Nutraceutical and Functional Food Market for the use of mashua anthocyanins not only as a source of natural colorants but also as a source of phytonutrients.     

Immunochemical and mass spectrometric analysis of Nε-(carboxymethyl)lysine content of AGE-BSA systems prepared with and without selected antiglycation agents

The present study was designed to compare surface plasmon resonance (SPR) biosensor, enzyme-linked immunosorbent assay (ELISA), and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods for the analysis of Nε-(carboxymethyl)lysine (CML) in glucose-bovine serum albumin (BSA) model systems and to investigate the possible inhibitory effect of selected compounds (α-tocopherol, ferulic acid, rutin, thiamin, thiamin monophosphate, and thiamin pyrophosphate) on CML formation. The reported levels of CML detected were dependent upon the method of analysis employed. The highest reported concentrations were obtained with the SPR biosensor, whereas the lowest were found by ELISA. However, a high correlation was observed between these two immunochemical procedures. CML concentrations were dependent upon the type and concentration of the candidate CML inhibitor. All inhibitory compounds investigated, with the exception of α-tocopherol, decreased the level of CML formation in the glucose-BSA system.     

Efficacy of commercial Fe(III)‐EDDHA and Fe(III)‐EDDHMA chelates to supply iron to sunflower and corn seedlings

Use of synthetic iron (Fe) chelates is the most common and effective way to treat Fe chlorosis in plants. Most commercial products contain Fe‐EDDHA or Fe‐EDDHMA but their efficacy can be quite different. Commercial products with EDDHA or EDDHMA as active components were chosen based on the data obtained by Lucena et al. (1992) in their chemical test. The chelates present extreme differences in behavior in the mentioned chemical tests. The analysis of the products revealed that the total Fe concentration is greater than the one indicated by the manufacturer in spite of a lesser amount of FeY^‐ present. The plant response to these commercial products was tested using short‐term greenhouse hydroponic cultures. Sunflower and corn were chosen because of their different behavior under Fe‐stress conditions. No significant difference between plants treated with Fe‐EDDHA or Fe‐EDDHMA chelates were observed. Since the purity index indicates there are too many differences between commercial formulations of the same type of chelate, the differentiation between groups cannot be determined with commercial products. Index I3, described by Lucena et al. (1992), does not correlate with the plant response because it did not consider the purity percentage of the products.     

Molecular Weight Distribution of (1→3)(1→4)-β-Glucan Affects Pasting Properties of Flour from Oat Lines with High and Typical Amounts of β-Glucan

Experimental (N979-5-2-4 and IA95111) and traditional oat lines (Jim and Paul) with average %β-glucan of 7.5, 7.8, 4.9, and 4.4%, respectively, were grown in 2002, 2003, and 2004. Molecular weight (MW) distributions of the β-glucans were examined for potential variations among growing years and for relationships with pasting properties measured by Rapid ViscoAnalyser under three separate conditions: 1) in silver nitrate (SN) solution to inactivate enzymes; 2) hydrolyzed by α-amylase to eliminate the effect of starch; and 3) treated with lichenase to remove β-glucan. The β-glucan was extracted by a process involving multiple precipitation and dialysis steps, and the MW distributions were determined by HPLC. %β-Glucan in N979-5-2-4 and IA95111 lines were consistently and significantly greater (P < 0.05) than in Jim and Paul lines during three growing years. The contribution of β-glucan to peak viscosity on the RVA was substantially greater than that of the starch for all three years. The molecular number average and peak MW of β-glucan from N979-5-2-4 and IA95111 were greater than these values for Jim and Paul, and values were consistent among years. The MW of extracted β-glucan was associated with pasting properties after amylase hydrolysis, but not after treatment with lichenase or in SN solution.     

Preliminary investigation for quali-quantitative characterization of soils contaminated with 241Am and 152Eu by low-altitude unmanned aerial vehicles (UAVs) equipped with small size γ-ray spectrometer: detection efficiency and minimum detectable activity (MDA) concentration assessment

Journal of Soils and Sediments - Localization and quali-quantitative characterization of radionuclide-contaminated soils are essential for healthcare and remediation activities. However,...     

Can commercial mulches be reservoirs of invasive earthworms? Promotion of ligninolytic enzyme activity and survival of Amynthas agrestis (Goto and Hatai, 1899)

Amynthas agrestis is an exotic, invasive earthworm in North America that has been associated with horticulture settings as well as damage to forest soil. An experiment was conducted to find out whether A. agrestis, an earthworm commonly found in mulches in Vermont, stimulates ligninolytic enzymes in the presence of commercial wood mulches. Mesocosms filled with a sandy loam soil were topped with either spruce, cedar or pine mulch. Half of the mesocosms received juvenile A. agrestis, the other half did not. After 7 weeks soils were analyzed for phenoloxidase and peroxidase activity. Most A. agrestis survived and developed into adults during the incubation period. Significantly greater phenoloxidase activity was detected in soils with A. agrestis than without earthworms. Mean (standard deviation) phenoloxidase activities in the presence of A. agrestis were 0.15 (±0.10), 1.14 (±0.46), 2.71 (±0.98) μmol g⁻¹ h⁻¹ for pine, spruce and cedar respectively, and 0.012 (±0.023), 0.25 (±0.25), 0.78 (±0.45) μmol g⁻¹ h⁻¹ in the absence of A. agrestis. There was significantly greater peroxidase activity for the pine and spruce treatment when earthworms were present. Mean peroxidase activities were 0.47 (±0.21), 0.94 (±0.29), 1.20 (±0.77) μmol h⁻¹ g⁻¹ soil for pine, spruce and cedar, respectively for soils with A. agrestis and 0.15 (±0.10), 0.37 (±0.10), 0.63 (±0.30) μmol h⁻¹ g⁻¹ soil in the absence of earthworms. The increased ligninolytic activity in combination with successful maturation of juveniles into adult A. agrestis suggests that mulch can be habitat for these invasive earthworms. This finding is supported by a survey of master gardeners in Vermont and New Hampshire 20% of whom reported to have seen these earthworms mainly in their gardens and mulched beds.     

Reaction of the butter flavorant diacetyl (2,3-butanedione) with N-α-acetylarginine: a model for epitope formation with pulmonary proteins in the etiology of obliterative bronchiolitis

The butter flavorant diacetyl (2,3-butanedione) is implicated in causing obliterative bronchiolitis in microwave popcorn plant workers. Because diacetyl modifies arginine residues, an immunological basis for its toxicity is under investigation. Reaction products of diacetyl with N-α-acetylarginine (AcArg) were determined as a model for hapten formation, with characterization by mass spectrometry, NMR, and HPLC with UV detection and radiodetection. Four products were identified by LC-MS, each with a positive ion of m/z 303 (diacetyl + AcArg); one pair displayed an additional ion at m/z 217 (AcArg), the other pair at m/z 285 (- H(2)O). Their (1)H-(13)C NMR correlation spectra were consistent with the addition of one or two of the guanidine nitrogens to form aminols. Open-chain pairs interconverted at pH 2, as did the cyclized, but all four interconverted at neutral pH. This is the first structural characterization of the covalent adducts between diacetyl and an arginine moiety.     

Physical and sensory properties of all-barley and all-oat breads with additional hydroxypropyl methylcellulose (HPMC) β-glucan

Hydroxypropyl methylcellulose (HPMC) is a substituted cellulose that reduces serum cholesterol at modest intake levels. HPMC has also been used for decades in gluten-free breads at a level to optimize loaf volume. Because consumers resist the consumption of whole wheat breads, the sensory and physical properties of all oat and barley breads incorporating HPMC were evaluated. Oat and barley also contain β-glucan, a glucose polymer similar to HPMC that also lowers cholesterol. The textural and sensory properties of the breads were determined by instrumental and chemical methods and sensory panels. HPMC increased the loaf volume of the breads by up to 2 times and decreased hardness immediately after baking and after up to 3 days of storage. Barley bread with HPMC was rated the highest in overall acceptability by sensory panelists compared to oat and wheat breads with or without HPMC.     

Prediction of Polycyclic Aromatic Hydrocarbon Bioaccessibility to Earthworms in Spiked Soils by Composite Extraction with Hydroxypropyl-β-Cyclodextrin and Organic Acids

Traditional exhaustive extraction methods often overestimate the risk of polycyclic aromatic hydrocarbon(PAH) bioaccessibility to biota. Therefore, reliable assessment methods need to be established. In this study, a composite extraction with hydroxypropyl-β-cyclodextrin(HPCD) and three low-molecular-weight organic acids, oxalic acid(OA), malic acid(MA), and citric acid(CA), was used to predict the PAH bioaccessibility to earthworms, subjecting to two soils(red soil and yellow soil) spiked with selected PAHs,phenanthrene, pyrene, chrysene, benzo(a)anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, and benzo(a)pyrene. For both soils,concentrations of PAHs by composite extraction using HPCD-OA(R~2= 0.89–0.92, slope = 1.89–2.03; n = 35), HPCD-MA(R~2=0.92–0.96, slope = 1.43–1.67; n = 35), and HPCD-CA(R~2= 0.92–0.96, slope = 1.26–1.56; n = 35) were significantly correlated with PAH accumulation in the Eisenia fetida earthworms. Moreover, the HPCD-CA-and HPCD-MA-extracted PAH concentrations were closer to the earthworm-accumulated PAH concentration than the extraction using just HPCD. The results indicated that the composite extraction could improve the prediction of PAH bioaccessibility, and therefore can serve as a reliable chemical method to predict PAH bioaccessibility to earthworms in contaminated soils.     

Effect of inhibitor compounds on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in model foods

The possible adverse effects on health of diet-derived advanced glycation endproducts (AGEs) and advanced lipoxidation endproducts (ALEs) is of current interest. This study had the objective of determining the effects of the addition of AGE/ALE inhibitors and different types of sugar and cooking oil on Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) formation in model foods (sponge cakes). The cake baked using glucose produced the highest level of CML (2.07±0.24 mmol/mol lysine), whereas the cake baked using fructose produced the highest concentration of CEL (25.1±0.15 mmol/mol lysine). There were no significant differences between CML concentrations formed in the cakes prepared using different types of cooking oil, but significant differences (P<0.001) were observed between the cakes prepared using different proportions of cooking oil. The cakes containing oil generated greater concentrations of CML than sucrose. α-Tocopherol and rutin did not inhibit CML and CEL formation. In contrast, ferulic acid and thiamin, thiamin monophosphate, and thiamin pyrophosphate reduced CML and CEL formation.     

Genotoxic Effects in Erythrocytes of Oreochromis niloticus Exposed to Nanograms-per-Liter Concentration of 17??-Estradiol (E2): An Assessment Using Micronucleus Test and Comet Assay

Pharmacologically active substances used in the treatment of human and animal illnesses may usually enter the aquatic environment via effluents from sewage treatment plants, as they are not completely biodegraded or removed during waste water treatment. 17??-Estradiol genotoxicity was evaluated in Oreochromis niloticus (family Cichlidae) using micronucleus test, other nuclear abnormalities assessment, and the comet assay with erythrocytes. Fish were exposed to aqueous systems contaminated with 6 ng/L 17??-estradiol for three periods: 24 h, 48 h, and 10 days. The results showed that 17??-estradiol has genotoxic potential in different periods, since significant differences (P?=?0.036) were observed in the micronucleus frequencies of the 10-day exposure groups in relation to the control group. Also, the same was observed when comparing the nuclear abnormality frequencies (P?=?0.018) of the 24-h exposure group with the negative control group, and when using comet assay (P?<?0.001) for 48-h evaluations. The tested concentration of the 17??-estradiol gave rise to mutagenic and genotoxic effects on the blood cells of O. niloticus, therefore the substance being considered a clastogenic chemical contaminant on both acute and chronic exposures. The assessment using a combination of micronucleus test, nuclear abnormalities, and comet assays proved to be suitable and useful in the genotoxicity testing of 17??-estradiol at nanograms per liter.     

Molecular and cytogenetic characterization of a collection of bahiagrass (Paspalum notatum Flügge) native to Uruguay

Paspalum notatum is a subtropical grass present throughout America, and one of the main constituents of the natural grasslands in Uruguay. An apomictic autotetraploid (2n?=?4x?=?40) is the most frequent cytotype. The occurrence of sexual diploids (2n?=?2x?=?20) has also been reported as well as the occasional presence of apomictic triploids and pentaploids in Argentina. In this study, ISSR (inter simple sequence repeats) molecular markers were used to analyze the genetic variability of 210 P. notatum individuals from a collection from Uruguay. Cytometric analyses and chromosome counts were used to assess the ploidy level of the individuals. All plants from Uruguay analyzed were tetraploid. Intra- and inter-population variability was found both in genomic DNA content and at the genotypic level. Several multilocus genotypes were shared among individuals within populations and among populations over moderate geographical ranges, at the same time, very dissimilar genotypes were found within the same population. Part of the genetic variance among populations can be explained by a broader scale geographic structure which is partly coincident with the traditionally recognized grassland management regions. In spite of the apparently high degree of genetic admixture within populations, groups of related genotypes seem to follow a broader geographical structure in the area under study. These results suggest that an efficient collection strategy for this apomictically reproducing species should include carefully planned intra- and inter-population sampling. A broader scale regional sampling strategy should also be considered although further studies will be required to define genetic structure at this level.     

Improved purity and immunostimulatory activity of β-(1→3)(1→6)-glucan from Pleurotus sajor-caju using cell wall-degrading enzymes

The objective of this work was to improve the purity of β-(1→3)(1→6)-glucan in the native triple helical structure from the fruiting bodies of Pleurotus sajor-caju for effective biological function using cell wall-degrading enzymes. A crude carbohydrate was extracted with hot water, then treated with crude xylanase and cellulase from Paenibacillus curdlanolyticus B-6. β-Glucan in the extract was purified to homogeneity with a single and symmetrical peak using 650M DEAE Toyopearl and Sepharose CL-6B column chromatography. The purity of β-glucan was confirmed by high-performance size-exclusion chromatography. Purified β-glucan was obtained at a purity of up to 90.2%. The Congo red reaction and atomic force microscopy indicated that the purified β-glucan exhibited a triple helix conformation. Purified β-glucan was able to effectively up-regulate the functions of macrophages such as nitric oxide (NO) and tumor necrosis factor (TNF-α) production.